The present invention relates to enzymatic hydrolysis of substances, particularly proteinaceous substances and more particularly, proteinaceous food substances.
Interest in the use of enzymes to hydrolyze substances has increased significantly in industry over the past twenty-five years, particularly in the food industry, since with acid hydrolysis, which has long been employed, some essential amino acids are entirely decomposed and others are partially decomposed. In addition, it now is recognized that during hydrolysis of proteins with hydrochloric acid, by-products, including such as what are known as chlorohydrins, i.e., chloropropanol and diol compounds, which may pose health concerns, are formed.
Enzymatic hydrolysis of substances operates on the basic theme of cleavage of chemical bonds of the substance. In general, preparation of hydrolysates intended to be employed themselves as a consumable comestible product, such as for nutritional purposes, or for other uses such as flavorants or for preparation of other products, or to be employed for obtaining a particular product fraction or fractions thereof for such or other uses, for example, is effected by incorporating an enzyme preparation with an aqueous suspension of a substance under conditions wherein, for process efficacy, the substance is at least partially solubilized. The selection of the enzyme preparation and other reagents is based upon the compositional chemical structure of the substance(s) to be hydrolyzed and by a desired hydrolysate product specification.
Use of sterile lactase enzyme preparations in pasteurized or sterilized milk and milk products to break down lactose, such as referred in Bijl, Canadian Patent No. 1 246 476 is known, and it is believed that sterile enzyme preparations have been employed in the pharmaceutical industry. However, generally in the context of hydrolyzing proteins, particularly in the industrial setting for preparation of nutritional and flavorant comestible products, microbiological contamination is generally not considered problematical, since the hydrolysis procedures are carried out generally at a temperature in a range of from 50.degree. C. to 60.degree. C. for from about 8 hrs to about 12 hrs so that, as noted by Eriksen, et al., PCT Patent Application Publication No WO 92/11771, bacterial growth is limited. Moreover, although it is reported in the art that enzymatic hydrolysis of proteins may be carried out at temperatures less than 50.degree. C., in general, even in such cases, microbiological contamination generally may not be of concern since the protein hydrolysate products are heated after preparation to a temperature and for a time at least sufficient to inactivate the enzymes, and use of temperatures and times to effect pasteurization or sterilization of the product are common.
On the other hand, however, it is noted that the age-old preparation of soy sauce from a koji avoids microbiological contamination with a high concentration of sodium chloride, but that, however, is not necessarily now deemed desirable for health reasons and is not, moreover, conducive for activities of many enzymes. Other agents to inhibit microorganism growth also have been employed, such as suggested by Kemmerer, U.S. Pat. No. 2,180,637, and such as employed by Kikuchi, et al., U.S. Pat. No. 3,857,967, but the agents disclosed are undesirable for food applications.
As compared with known microorganism fermentations or acid hydrolysis procedures, which have a limited capacity for tailoring the resultant products, since, generally, only the extent, or degree, of hydrolysis can be controlled readily, enzymatic hydrolysis theoretically enables, with regard to any particular substrate treated, obtaining a variety of products tailored to particular specifications with greater precision. However, even to the extent that such may be the case, as is documented throughout the art, in general particularly in terms of a cost/benefit analysis, although dependent upon the quantity of enzyme employed, yields of desired products obtained by enzymatically hydrolyzing proteins are considered low, and it is generally the rule, rather than the exception, that such procedures result in substantial amounts of by-product(s) for which uses are relatively few and not of great economic value.
A factor which impacts upon yield of desired product(s) is that enzyme preparations produced for general industrial use, known in the art as commercial-grade enzymes, generally are a mixture, or "cocktail", of a plurality of enzymes which differ in substrate affinity and specificity, hereinafter "activity", i.e., the capability of effecting a particular substrate chemical bond cleavage under any particular set of reaction conditions, including pH and temperature. Although the activity of one enzyme of the preparation cocktail generally is predominant, and although situations are known wherein two desirable activities of the enzyme preparation may be sequenced such as by pH control, as disclosed in Gianna, et al., European Patent Application Publication No. 0 320 717, the other enzyme(s) of the preparation, which may be considered "impurities", may produce results and effects which may be competitive with, or even at odds with, the desired effects and results. For example, depending upon the substrate and/or the conditions of hydrolysis, the "impurities" may detract from a theoretical yield because such may induce competitive reactions and/or even may be destructive of the enzyme having the predominant activity, thus inducing reaction inhibition beyond that which would be expected, based upon theoretical considerations.
To enhance process control and final product specificity, use of substantially pure enzymes would be desirable. However, the attendant increased enzyme preparation cost due to purification procedures generally presents a cost/benefit ratio which generally can not be justified in general industrial use, other than such as in the pharmaceutical industry, or for relatively small-scale high-value analytical purposes or bio-engineering. Such is particularly the case in the food flavorant art, particularly when the cost/benefit of purified enzyme use is compared to that of carrying out conventional microorganism fermentations or an acid hydrolysis.
To address the afore-noted problems, it is not uncommon in industrial practice, particularly in the comestible, i.e., food, art, to employ greater amounts of commercial-grade preparations to amplify the activity of the dominant enzyme of a given preparation, as compared with what theoretically would be required for any given set of processing conditions, and to carry out the reaction for about 8 hrs to about 12 hrs. For example, employing a greater amount of enzyme preparation speeds up the reaction rate for any given set of conditions, and in general, when subjecting proteinaceous substances to a moderate degree of hydrolysis to obtain a hydrolysate which has a broad peptide profile and which is comprised of constituents having a molecular weight in excess of 10,000 Daltons, difficulties are not encountered operating in this manner.
Various problems arise, however, when it is desired to obtain a protein hydrolysate, such as one suitable for nutritive applications, which has a high degree of hydrolysis so that the product contains a significant amount of free amino acids and/or a narrow peptide size-range profile, e.g., a molecular weight below about 10,000 Daltons and preferably, below about 6,000 Daltons. As is discussed in the art, such products are useful in a wide variety of food and nutritive applications, including formulas for infants allergic to milk proteins. However, unless particular reactant combinations and conditions are employed, such as in Jost, U.S. Pat. No. 5,039,532, yields of such products, as indicated above, generally are considered undesirably low, and in general, such procedures are considered to be expensive because of the enzyme preparation(s) employed. On the other hand, to reduce enzyme usage, the substrate employed may be dilute, which likewise makes process economics unattractive because of unit/volume considerations.
To obtain high-value end-use products as noted above, it also is known to combine enzymatic procedures with hydrolysate isolation procedures such as ultrafiltration. Such procedures include those disclosed in Eriksen, et al., PCT Patent Application Publication No. WO 92/11771, and in Nielsen, et al., PCT Patent Application Publication No. WO 93/24020, for example, and an alternative to separate isolation procedures has been proposed in Maubois, et al., U.S. Pat. No. 4,427,658, which discloses to employ an ultrafiltration membrane reactor, which may be used in a continuous mode, to recycle and further process a permeate. Maubois, however, indicates that the ratio of enzyme concentration to protein concentration must be on the order of 8% to 15%.
Thus, it long has been and still is desired in the food industry, particularly in the context of hydrolyzing proteins, to obtain, consistently, enzymatic hydrolysates of low molecular weight and/or of narrow peptide profile and to solve the dichotomy of how to increase yields while at the same time reducing the amount of enzyme usage to achieve cost savings and cost/benefit effectiveness.